Anthraquinone and its derivatives

ABSTRACT

There is disclosed a compound of formula (I) wherein each of X 1  and X 2  are independently NH-A-NR 1 R 2 , and wherein A is A C 2-8  alkylene and R 1  and R 2  are independently selected from hydrogen, C 1-4  alkyl, C 2-4  hydroxy-alkyl and C 2-4  aminoalkyl, or R 1  and R 2  together form a C 2-6  alkylene group which with the nitrogen atom to which R 1  and R 2  are attached forms a heterocyclic ring, or an N-oxide derivative thereof, and wherein the compound (I) or its N-oxide derivative is optionally in the form of an acid salt derived from an organic or inorganic acid. Also disclosed is a method of its production and its uses, including its use in analyzing a cell or biological material and detecting the emitted fluorescence signal.

This invention relates to an anthraguinone and its derivatives, inparticular, although not exclusively, including its applications in arange of fluorescence detection technologies.

There are a number of DNA-binding fluorochromes available which coverthe UV and visible region of the spectrum. Recently, very brightDNA-intercalating cyanine fluorochromes, based upon modified dimers ofthiazole orange, have become commercially available. These cyanine dyesdo not share the cell permeant properties of other DNA specificUV-activated fluorochromes. Furthermore, the commonly usedDNA-interactive fluorochromes have fluorescent signatures which overlapthose of other fluorochromes, activated in the spectral range of visiblelight, which are used as molecular tags to probe aspects of cell biologyor biological structures. Examples of currently known cyanine dyes aredisclosed in U.S. Pat. Nos. 5,410,030 and 5,436,134.

The present invention seeks to develop cell permeant DNA-interactiveagents which may provide a fluorescence signature extending in to theinfra red region of the spectrum. Such an agent could, for example, beoptimally excited by red-line emitting lasers inmulti-laser/multi-fluorochrome applications for both fixed specimens andviable cells.

Thus, in accordance with a first aspect of the present invention, thereis provided a compound of the following formula (I)

wherein each of X₁ and X₂ are independently NH-A-NR¹R², and wherein A isa C₂₋₆ alkylene group and R¹ and R² are independently selected fromhydrogen, C₁₋₄ alkyl, C₂₋₄ hydroxyalkyl and C₂₋₄ aminoalkyl, or R¹ andR² together form a C₂₋₆ alkylene group which with the nitrogen atom towhich R¹ and R² are attached forms a heterocyclic ring,

-   -   or an N-oxide derivative thereof,    -   and wherein the compound (I) or its N-oxide derivative is        optionally in the form of an acid salt derived from an organic        or inorganic acid.

The term “alkylene” here is used to mean an alkyl chain.

In a preferred embodiment, when R¹ and R² form a heterocyclic ring, thering has 3 to 7 carbon atoms therein preferably, both X₁ and X₂ are bothNH(CH₂)₂NR¹R². In particular, it is preferred that R¹ and R² are bothC₁₋₄ alkyl groups, preferably methyl groups.

According to a second aspect of the present invention, there is provideda compound of the following formula (II):

In one embodiment, compound (II) may be in the form of its N-oxidederivative.

The compound of the general formula (I) and, in particular the specificcompound (II) may be used as, for example, a DNA dye and may be a puresynthetic compound which is soluble in biologically compatible solventsincluding water. Compound (II) has a high infinity for DNA (the DNAbinding constant is approximately 10e7 M-1) and has the capacity toenter living cells rapidly.

The absorbance spectrum for compound (II) shows Ex_(λmax) near 647 nmand produces a fluorescence spectrum extending from 665 nm out to beyond780 nm wavelengths (Em_(λ) _(max) is about 677.5 nm)

According to a further aspect of the present invention, there isprovided a method of preparing a compound of the following formula (I):

wherein each of X₁ and X₂ are independently NH-A-NR¹R², and wherein A isa C₂₋₈ alkylene group and R¹ and R² are independently selected fromhydrogen, C₁₋₄ alkyl, C₂₋₄ hydroxyalkyl and C₂₋₄ aminoalkyl, or R¹ andR² together form a C₂₋₆ alkylene group which with the nitrogen atom towhich R¹ and R² are attached forms a heterocyclic ring,

-   -   or an N-oxide derivative thereof, and wherein the compound (I)        or its N-oxide derivative is optionally in the form of an acid        salt derived from an organic or inorganic acid,

the method comprising the step of reacting a compound of the followingformula (III)

with NH₂-A-NR¹R²R wherein A, R¹ and R² are as defined above.

The method preferably further comprises the step of treating theresultant compound with an acid, preferably concentrated sulphuric acid.In addition, in a preferred embodiment, the method may further comprisesubsequent treatment with sodium chlorate and/or sodium hydrogensulphite.

Modelling has demonstrated that the compounds of the present inventioncan form stable, intercalated complexes with DNA. Thus, according to afurther aspect of the present invention, there is provided a fluorescentcomplex comprising a nucleic acid and a compound of the followingformula (I):

wherein each of X₁ and X₂ are independently NH-A-NR¹R², and wherein A isa C₂₋₈ alkylene group and R¹ and R² are independently selected fromhydrogen, C₁₋₄ alkyl, C₂₋₄ hydroxyalkyl and C₂₋₄ aminoalkyl, or R¹ andR² together form a C₂₋₆ alkylene group which with the nitrogen atom towhich R¹ and R² are attached forms a heterocyclic ring,

-   -   or an N-oxide derivative thereof,    -   and wherein the compound (I) or its N-oxide derivative is        optionally in the form of an acid salt derived from an organic        or inorganic acid.

The nucleic acid is preferably DNA. It has been found that the DNA maybe present in a living cell. The compounds of the present invention maystain fixed human chromosomes. As the DNA:Compound molar ratio increasesthere is a bathochromic shift in the compound plus DNA solutionspectrum. At high DNA:Compound ratios, attainable within living cells,the spectral shift contributes to an already significant separation ofthe compound-DNA emission spectrum from that of an example of ared-fluorescing compound Cy 5.

According to a further aspect of the present invention, there isprovided a method of analysing a cell or biological material containingone or more nucleic acids, comprising the steps of:

-   -   a) preparing a biologically compatible solution containing a        compound of the formula (I):        wherein each of X₁ and X₂ are independently NH-A-NR¹R², and        wherein A is a C₂₋₈ alkylene group and R¹ and R² are        independently selected from hydrogen, C₁₋₄ alkyl, C₂₋₄        hydroxyalkyl and C₂₋₄ aminoalkyl, or R¹ and R² together form        C₂₋₆ alkylene group which with the nitrogen atom to which R¹ and        R² are attached forms a heterocyclic ring,    -   or an N-oxide derivative thereof, and wherein the compound (I)        or its N-oxide derivative is optionally in the form of an acid        salt derived from an organic or inorganic acid;    -   b) treating the cell or biological material with the        biologically compatible solution;    -   c) exciting the compound (I) in the treated cell or biological        material with a light source; and    -   d) detecting the emitted fluorescence signal.

The compound of formula (I) may be present in its free state or becomplexed to other molecule (s), for example either by covalent ornon-covalent attachment.

The light source preferably provides wavelength(s) in the spectralregion of the wavelength(s) of maximum absorption of compound (I).

It has been found that the fluorescence signature of the compounds ofthe present invention extends to the infra red region of the spectrum.The compound of the present invention may be present in the cell orbiological material in combination with one or more other fluorochromesor light-emitting compounds. The other fluorochromes may emit in the UVor visible region of the spectrum. Thus, the compounds of the presentinvention lend themselves to multiparameter analysis with otherfluorochromes with spectra which overlap with those of the commonly usedvisible-region DNA probes.

The one or more other compounds may be used, for example, to detectAnnexin V and is preferably used in combination with the N-oxidederivative of compound (I). Flow cytometric analysis, for example withthe instrument in dual laser mode, may be used. The invention thus mayprovide a way of discriminating intact viable cells from thoseundergoing the various stages of cell death.

Thus, the compounds of the present invention provide far red/infra redfluorescent permeant DNA dyes suitable for cellular DNA analysis whereintact cells may be required, for example the detection of moleculeseither on the cell surface (e.g. a receptor molecule or marker fordifferentiation) or within cells (e.g. cytosolic enzymes) by methodswhich require the maintenance of membrane integrity to preventperturbation or loss of such molecules.

As mentioned above, in this method, the compounds of the presentinvention may stain nucleic acids in fixed human chromosomes, fixedcells and fixed biological materials, and in procedures which modify thepermeability of living cell membranes.

According to a further aspect of the present invention, there isprovided the use of compound (I) in a biological assay. Compound (I) maybe present either in its free state or complexed to other molecules byeither covalent or non-covalent attachment in the biological assay.Compound (I) may be present as an N-oxide derivative thereof. Thebiological assay is preferably a rapid and/or large capacity handlingprocedure. The use of the compounds of the present invention, asindicated by compound (I), as a discriminating or orientating parameterfor cell nuclei has been demonstrated for both flow cytometry andconfocal laser scanning microscopy.

In accordance with a further aspect of the present invention, there isprovided the use of compound (I) in cytometry. Compound (I) isoptionally present as an N-oxide derivative thereof. The cytometryprocess may be, for example, single beam or multi-beam flow cytometry.

By way of example, single beam (488 nm) flow cytometry has been used todemonstrate the utility of compound (I)-nuclear DNA fluorescence(preferably compound (II)-nuclear DNA fluorescence) as a discriminatingparameter for human blood and lymphoma cells, in combination withfluorochrome-labelled antibodies for the detection of surface antigensand subpopulation recognition. Compound (I) fluorescences was found toreflect cellular DNA content as evidenced by cell cycle DNA distributionprofiles for exponentially proliferating cell populations showing asteady-state or asynchronous distribution of cells with respect to cellcycle age, or for perturbed cell populations in which, for example, drugaction has caused the delay or arrest of cells at a given point in thecell cycle. In one embodiment, dual beam (488 nm/633 nm) flow cytometryshows the selective excitation of compound (I), preferably compound(II), and fluorescein in intact cells. In addition, in one embodiment,the application of compound (I), preferably compound (II), in triplebeam flow cytometry (multiline UV/488 nm/G33 nm) has been demonstratedin applications involving delayed signal discrimination where beamseparation allows for the discrimination of the excitation beamassociated with a fluorescence emission signal by reference to the delayin signal arrival at a detector.

According to a further aspect of the present invention, there isprovided the use of compound (I) in microscopy compound (I) may bepresent as its N-oxide derivative. Preferably the microscopy is confocallaser scanning microscopy (CLSM). By way of example, CLSM employingeither is 647 nm or 568 nm wavelength excitation of intracellularcompound (I), preferably intracellular compound (II), shows fluorescencespecifically located in the nucleus revealing nuclear architecturewithin living or fixed human cells.

According to a further aspect of the present invention, there isprovided the use of compound (I) as a nuclear-staining agent. Compound(I) may be present as its N-oxide derivative.

According to a further aspect of the present invention, there isprovided the use of compound (I) as an imaging agent. Compound (I) maybe present as its N-oxide derivative.

In one embodiment, compound (I) can be used as an imaging agent inmulti-photon excitation imaging.

Dual wavelength imaging, using compound (I) to reveal nuclear form, maybe used to demonstrate the heterogeneity in esterase-dependentfluorescein loading, of whole cells and in the assessment ofmitochondrial function by rhodamine 123 labelling. In such imagingapplications, compound (I) shows no evidence of photo bleaching and waspersistent.

Thus, the compounds of the present invention can be considered as afluorochrome for application as an agent in the use, calibration,standardization, and configuration of fluorescence-based systems. Thepreferred compound of the present invention is compound (II)-deep redfluorescing bisalkylaminoanthraquinone (DRAQ5).

It has been found that the high penetration of red line laser beams intotissues and the permeant properties of the compounds of the presentinvention provide a combination which allows three dimensionalorientation and location of nuclei within living tissues. In addition,the availability of low cost HeNe lasers or other red light-emittingdevices with enhanced power enables the compounds of the presentinvention to find applications in detection systems where theirfluorescence signature can be used as a discriminating parameter.

Whilst the invention has been described above, it extends to anyinventive combination of the features set out above or in the followingdescription.

The invention will now be described, by way of example, with referenceto the accompanying drawings and examples, and in which:

FIG. 1 a to c show spectral characteristics of DRAQ5;

FIGS. 2 a to f show spectral characteristics of DRAQ5 associated DNAfluorescence detected by CLSM;

FIG. 2 g shows the multi-photon imaging of DRAQ5 stained cell nuclei;

FIG. 2 h to k shows a comparison of viable cells stained by DRAQ5 or itsN-oxide derivative (DRAQ5N);

FIGS. 3 a to d show differential excitation of fluorescein and DRAQ5 inviable A375 cells analysed by CLSM;

FIGS. 4 a to c show the differential excitation of rhodamine 123 andDRAQ5 in viable A375 cells analysed by confocal laser scanningmicroscopy;

FIG. 5 shows flow cytometric analyses of DRAQ5 accumulation, for a onehour exposure period, in viable HL60 cells;

FIGS. 6 a to d show dual beam flow cytometric analysis for the detectionof DRAQ5-associated fluorescence in fluorescein-labelled viable HL60cells;

FIGS. 7 a to d show single beam flow cytometric analysis of DRAQ5fluorescence versus antibody fluorescence7 for cultured andblood-derived human cells;

FIG. 8 shows single beam flow cytometric Quantification of fluorescenceintensity of cultured and blood-derived human cells exposed to DRAQ5;

FIG. 9 shows dual beam flow cytometric analysis of the cell cyclespecific expression of cyclin BE;

FIGS. 10 a to f show triple beam flow cytometric analysis ofDRAQ5-stained fixed and RNaseA digested asynchronous SUD4 lymphomacells;

FIG. 11 a to c show the flow cytometric analysis of cellular DNA contentof intact SUD4 lymphoma cells using 488 nm, 633 nm or multi-line UVexcitation;

FIGS. 12 a-d illustrate examples of cellular accumulation, using a humanB cell lymphoma cell line, using combinations of reagent treatments; and

FIGS. 13 a-d illustrate examples showing the same combination ofreagents for VP-16 treated cultures.

EXAMPLE 1 Synthesis of DRAQ5

Procedure:

1,5-dichloroanthrocuinone (15 g, 54 mmoles) was dissolved inN,N-dimethylethylenediamine (47.6 g, 540 mmoles) are refluxed for 18h.The reaction was monitored by TLC (9:1 CH₂Cl₂/MeOH). The mixture wascooled to room temperature and diluted with water to precipitate thetitled-compound. The filtered solid was recrystallised from methanol toafford (A) (15-89, 89%) as a crystalline solid. R_(f) (9:1CH₂Cl₂/MeOH):0.60.

¹H NMR (CDCl₃): δ 9.8 (t,2H), 7.6 (m,4H), 6.9 (m,2H) 3.4 (q, 4H), 2.7(t, 4H), 2.4 (5, 12H)

Mass spectrum, m/z 381 (m⁺+1).

The anthracene-9,10-dione derivative (A) (6 g, 15.8 mmoles was dissolvedin 65 g of concentrated H₂SO₄ and cooled to −10° C. Anhydrous sodiumchlorate (6.5 g, 61.6 mmoles) was added in portions over 1.5 h and themixture then stirred for 3 h at room temperature. The blue solution wasadded slowly to a cold sodium hydrogen sulfite solution (1%, 1000 ml).The mixture was neutralised to pH7 with 5M NaOH. The titled compound (B)was extracted from the aqueous phrase with CH₂Cl₂ and concentrated undervacuo. Column chromatography (SiO₂, 9:1 CH₂Cl₂/MeOH) gave (B) (1.2 g,20%)

EXAMPLE 2 Synthesis of DRAQ5N[1,5-Bis-((2-dimethylamino-N-oxide)ethyl)amino)-4,8-dihydroxyanthracene-9,10-dione]

The title compound was prepared from example 1 (DRAQ5) as follows. DRAQ5(0.19, 24 mmol) was added to meta-chloroderoxybenzoic acid (80% purity,0.186 g, 0.96 mmol) in dry dichloromethane and left at −20° C.overnight. The crude product was subjected to silica columnchromatography using 9:1:0.1 dichloromethane:methanol:ammonia (0.88sp.gravity) as an eluting solvent. The title compound was isolated as ablue powder. Melting Point 221° C.1H NMR (CD3CD) d (delta) 7.39 (d, 2H),7.2 (d, 2H), 4.0 (t, 4H), 3.65 (t, 4H) 3.30 (S, 12H) 13C NMR (CD3OD): d(delta) 189, 156.5, 147, 130, 122.5, 116, 69.5, 59.5, 38.5. Massspectrum m/z 445(M++1).

EXAMPLE 3 Spectral Analysis of DRAQ5

Absorbance spectra were obtained using a Perkin-Elmer Lambda 16 UVspectrometer and a 10 μM solution of agent dissolved in dichloromethaneand measured in a 1 cm path length quartz-silica cuvette. Fluorescencespectra for a 0.8 ml solution of 20 μM DRAQ5 in a 1 cm path lengthsemimicro quartz silica cuvette were determined by exciting at 647 nmwavelength or monitoring emission at 670 nm wavelength. Fluorescencemeasurements were made on a Perkin Elmer LS50 spectrofluorometer withslit widths set at 10 nm. The spectrofluorometer was equipped with ared-sensitive photomultiplier tube (PMT; type R928; Hamamatsu PhotonicsKK, Japan). Data were accumulated for four scans for each condition andexported into a spreadsheet program to correct values for the buffercontrol and to determine emission maxima. DNA-DRAQ5 fluorescence wasmeasured by the addition of microlitre volumes of concentrated calfthymus DNA solutions to the cuvette with mixing. Both agent and DNA wereprepared in DNA binding buffer (0.05 M sodium phosphate, pH 6.2, 0.05 MNaCl, 0.001 M EDTA; 3). The spectra shown were corrected for the bufferbackground and not for the spectral sensitivity of the PMT. Rhodamine123 spectra were generated in DNA binding buffer using either 488/5 nmexcitation or monitoring emission at 530/5 nm. Previously publishedexcitation and emission spectra, were obtained from original sourcefiles and normalised for peak intensity.

Spectral Characteristics and Interaction of DRAQ5 with DNA

FIGS. 1 a-c show spectral characteristics of DRAQ5.

FIG. 1 a: Visible absorbance spectrum for DRAQ5 (10 μM indichloromethane).

FIG. 1 b, Comparison of excitation spectra for specified emissionwavelengths for: FITC (∘, 620 nm emission), rhodamine 123 (∇, 0.5 μg/ml,530 nm emission), Texas Red (Δ; 660 nm emission), Cy 5.18 (□, 715 nmemission), DRAQ5 (●, 670 nm emission)

FIG. 1 c: Comparative emission spectra for specified excitationwavelengths for FITC (∘, 425 nm excitation), rhodamine 123 (∇, 0.5μg/ml, 488 nm excitation), Texas Red (Δ, 500 nm excitation), Cy 5.18 (□,570 nm excitation), 20 μM-DRAQ5 (●, 647 nm excitation), and 20 μM DRAQ5plus 1280 μM DNA (●, 647 nm excitation).

FIG. 1 a shows the visible absorbance spectrum for DRAQ5 in phosphatebuffer at pH 7.4. The spectrum gave maxima at 622 and 676nm, in additionto maxima (data not shown) at 240 nm and 314 nm. The extinctioncoefficient at 676 nm wavelength was determined as 20949 cm⁻¹mol⁻¹. Thefluorescence characteristics of DRAQ5 were studied to permit theinterpretation of fluorometric data generated by flow cytometry andconfocal imaging. An excitation spectrum was generated for the 460-660nm range for emission at 680 nm wavelength and compared with oneoptimised for rhodamine 123 and those for other fluorochromes. FIG. 1 bshows that DRAQ5 excitation in the 630-650 nm region is essentiallysimilar to the excitation spectrum of the cyanine dye Cy 5.18 (EX_(λmax)649 nm) but distinct from that of Texas Red (Ex_(λmax) 596 nm),rhodamine 123 (Ex_(λmax) 511 nm) and fluorescein isothiocyanate (FITC;Ex_(λmax) 490 nm). In all cases shown in FIG. 1, spectra have beennormalised to the intensity values at either the Ex_(λmax) or Em_(λ)_(max).

The emission spectrum of DRAQ5 alone (FIG. 1 c) showed that for 647 nmexcitation there is significant emission extending from 665 nm out tobeyond 780 nm wavelengths with an Em_(λmax) of 677.5 nm. The emissionspectrum is significantly red-shifted compared with that of Cy 5.18.DRAQ5 appears to shows residual excitability at much lower wavelengthsalthough fluorescence intensity for 514 nm wavelength excitation wasreduced for DRAQ5 when compared with the values for excitation at 647nm, in keeping with the characteristics of the excitation spectrum (datanot shown).

Molecular modelling suggests that DRAQ5 is capable of binding to DNAthrough intercalation, the side chains on opposing sides of the aromaticring structure each having the potential to stabilise the molecule onDNA.

Fluorometric experiments indicate that DNA affects DRAQ5 fluorescence ina complex manner with increasing DNA:DRAQ5 ratios associated with a redshift of Em_(λmax) to 697 nm at a molar DNA:DRAQ5 ratio of 64. Thisshift upon DNA interaction is shown in FIG. 1 c. At high DNA:DRAQ5ratios, equivalent to those encountered in vital cell staining, loss ofDRAQ5 signal due to any dye-dye Quenching effects appears to be minimal.The red shift of Em_(λmax) and the considerable low infra red/infra redsignal at wavelengths beyond 730 nm distinguishes this probe from Cy5.18 despite similar excitation characteristics.

EXAMPLE 4 Imaging and Microscopy Applications of DRAQ5 as a Novel DeerRed/Infra Red Fluorescent DNA-Binding Probe

Preferred aspects of the invention relate to the development of a cellpermeant DNA-interactive dye, capable of acting as a discriminating ororienting marker for cellular DNA, with a fluorescence signatureextending into the infra red region of the spectrum. The inventionpermits multi-laser, multi-fluorochrome and multi-photon excitationmicroscopy methods to be used with both fixed specimens and viablecells. Here we describe the spectral characteristics of DRAQ5 anddemonstrate the potential applications of this DNA probe formultiparameter analysis of living and fixed cells using confocal laserscanning microscopy.

Cell Culture

The human melanoma cell line A375 was grown as asynchronous cultures inEagle's minimum essential medium supplemented with 10% foetal calfserum, 1 mM glutamrine and antibiotics and incubated at 37° C. in anatmosphere of 5% CO₂ in air. For imaging experiments, cells were grownat a density of 5×10⁴ cells/well as a monolayer on autoclaved glasscoverslips in 6-well plates for 48 h prior to treatment. Attached viablecells were mounted in fresh PBS for microscopy. Where indicated,attached cells were fixed with 70% methanol at −20° C. for 10 min priorto rehydration and staining with ethidium bromide at 5 μg/ml for 10 minin the presence of 5 mg/ml RNase A.

Drug Preparation and Treatment

DRAQ5 was synthesised using the principles described and stored at +4°C. as an aqueous stock solution of 10 mM. DRAQ5 dilutions were preparedin phosphate buffered saline (PBS) and added directly to cultures.Fluorescein diacetate (FDA; Koch Light Laboratories) was prepared as astock solution of 12 mM in acetone and stored at −20° C. Cells weretreated with 0.2 μM FDA for 10 min at 37° C. either alone or after a 50min exposure to DRAQ5. Likewise DRAQ5-treated cells were labelled withrhodamine 123 (laser grade; Kodak) at 2 μg/ml culture medium for 10 min,prior to analysis.

Confocal Laser Scanning Microscony (CLSM) of Intact Cells

The system used was a Leica TCS 4D (LaserTechnik Gmbh, Germany) scannercoupled to a Leitz DM R microscope and operating with an Ominchromeargon/krypton laser. The laser provided emission lines at 488, 568 and647 nm with variable power. Coverslip cultures were washed briefly inPBS, mounted in inverted positions on glass slides, the coverslips beingsupported at the edges by a piping of petroleum jelly to prevent thecells from being compressed. The slides were examined immediately using×100 or ×40 oil immersion objective lenses with mid-range pinhole andphotomultiplier gain settings. Excitation/emission wavelengths forDRAQ5, fluorescein and rhodamine 123 were 647 nm/>665 nm, 488 nm/>515 nmand 488 nm/>590 nm respectively. Gain settings were adjusted such thatthe most fluorescent drug-treated sample gave pixel intensities justbelow saturation. The black level/offset was adjusted to giveeffectively zero background (<4 for pixel value) after 16× line noisefiltration of images for untreated controls. Using this approach, theuntreated controls showed minimal autofluorescence and gave nodiscernible image obviating the need for a background correction. Savedimages were converted for analysis and merging using IP Lab SpectrumImage analysis software (Signal Analytics Corp. Vienna, Va., USA).

CLSM Analysis of DRAQ5 Fluorescence in Viable Cells.

To gain some insight into the dependence of DRAQ5 fluorescence on theexcitation wavelength and the spectral separation of its fluorescencesignal from that of another DNA probe, we have compared cells stainedwith either DRAQ5 or ethidium bromide. The sensitivity range of the CLSMat either 488 nm or 568 nm excitation was optimised with respect tofluorescence of ethanol-fixed cells stained with ethidium bromide (FIG.2 d-f), while imaging at 647 nm excitation was optimised onDRAQ5-treated viable cells (FIG. 2 c). FIG. 2 a-f shows that atfluorochrome concentrations adequate for imaging nuclei and withappropriate emission filtration, 488 nm and 647 nm excitation conditionscan be used to exclusively image either ethidium bromide or DRAQ5staining respectively. DRAQ5 could also be used to image fixed cellswith retention of much of the nuclear architecture observable in intact,viable cells (data not shown) Fluorescence activation has also beenobserved using multiphoton excitation of fixed cells stained with DRAQ5(human B cell lymphoma cells; ethanol fixed; 20 μM DRAQ5; YLF modelockedlaser excitation at 15 mW using a modified MRC600 confocal imagingsystem; Ex_(λ)=1047 nm; Em_(λ)=far red; FIG. 2 g).

Using CLSM with 647 nm excitation (FIG. 2 c) there was cleardemonstration of nuclear-located fluorescence, quite different fromother anthraquinone- and anthracycline-based7 agents screened whichproduced both nuclear and cytoplasmic signals. FIG. 2 c shows thatDRAQ5-treated viable cells display clear definition of nucleararchitecture and the definition of the edges of nucleolar and nuclearmembrane regions.

Thus, FIGS. 2 a-f show spectral characteristics of DRAQ5-associated DNAfluorescence detected by CLSM: Panels a-c, excitation at 488, 568 and647 nm wavelengths respectively for viable human A375 melanoma cells.Panels d-f, excitation at 488; 568 and 647 nm wavelengths respectivelyfor ethanol-fixed cells stained with ethidium bromide. Images are100×100 μm.

Multi-Photon Imaging of DRAQ5

The principle of 2-photon excited fluorescence microscopy was firstdemonstrated by Webb and co-workers (Science, 248, 73-76 (1990); U.S.Pat. No. 5,034,613). In essence this involves the capture of two photonsby an excitable molecule by arranging excitation conditions which favoursuch events. The excitation spectrum for a given fluorochrome formulti-photon events differs from the corresponding single photonexcitation spectrum although the emission spectra are independent of theexcitation mode. The key component of the excitation system, as appliedto imaging, is a tuneable or fixed wavelength mode-locked laser, givingultra-short pulses at high repetition rate. The multi-photon microscopetypically incorporates a tuneable Ti-Sapphire laser emitting within thewavelength-range 700-950 nm, with pulse widths of approximately 100femto-seconds, and a repetition rate of 80 MHz. Fixed wavelength laserscan also be used such as a YLF mode-locked laser providing multi-photonexcitation at 1047 nm. The peak intensity of such lasers is so high thatdye excitation can occur by absorption of two or more photons in rapidsuccession. Importantly, multi-photon excitation avoids the need forshort (e.g. UV) excitation wavelengths. Furthermore, since fluorescenceexcitation is localized to the region of the focal spot the multi-photonsystem can optically section a scanned object with restricted bleaching.Multi (dual) photon excitation of DRAQ5 has been achieved using YLFmode-locked laser and an example of a collected image showingnuclear-located fluorescence in fixed cells is shown in FIG. 2 g. Wehave also observed multi-photon excitation of DRAQ5 in the nuclei offixed cells using a Ti-Sapphire laser (pumped with 5W) emitting at 740nm wavelength (consistent with the ability to UV excite DRAQ5-treatedcell nuclei, as shown in FIG. 11). It is expected that the excitationspectrum for DRAQ5, consistent with the findings for otherfluorochromes, differs from that determined by single-photonspectroscopy. The penetrance of infra red laser beams offersapplications for multiphoton excitation of DRAQ5 in deep section/tissuescanning for nuclei location, quantification and morphology permittingaccurate 3D reconstruction of complex cellular environments.

FIG. 2 g shows the multi-photon imaging of DRAQ5 stained cell nuclei.Human B cell lymphoma cells were fixed with ethanol and stained with 20μM DRAQ5. YLF mode-locked laser excitation at 15 mW (Ex λ=1047 nm; Emλ=far red) was used and images gained using 60× N.A. 1.4 oil objective,a zoom factor of 1.9 and a Kalman averaging of 37 frames.

CLSM Analysis of Fluorescence of DRAQ5 and an N-oxide Derivative(DRAQ5N) in Viable Cells

We have sought to exemplify the effect of changes to the structure of acompound of the general form of compound (I) on viable cell stainingcharacteristics. An N-oxide derivative of DRAQ5 (ie DRAQ5N) retains thegeneral structure (I) but has lost overall charge. The change affectsthe efficiency of the binding potential of the agent in viable cellswhile retaining fluorescence, cell-permeant properties and nuclearlocation. FIG. 2 h to k show that under equivalent conditions for thedetection of nuclear flurorescence in viable human cells, DRAQ5integrated nuclear fluorescence intensity per nucleus section wasapproximately 10-fold greater than the value derived for DRAQ5N-treatedcells. Previous publications (see references 1-10 below) have describedthe characteristics of alkylaminoanthraguinone N-oxides and theirpotential as bioreductive pro-drugs. Thus the N-oxide of DRAQ5 (ieDRAQ5N) described here will share the properties of this class of agentsin being capable of bioreductive conversion to DRAQ5. We suggest thatthe novel fluorescence characteristics of DRAQ5 will provide a markerfor cellular bioreductive activity, and by implication hypoxic status,by virtue of DRAQ5N conversion. Thus, the present invention envisagesthe use of DRAQ5N as a marker for hypoxic cells.

Thus, FIG. 2 h to k show simultaneous CLSM capture of transmission(panels h and j) and the corresponding far red/low infra-redfluorescence (panels i and j respectively) images of viable HL60 cellsexposed to either 10 μM DRAQ5 or 10 μM DRAQ5N for 1 h.

-   1. Patterson L H: Anthraquinone anticancer compounds with    (disubstituted amino-N-oxide) alkylamino substituent. UK Patent    GB2237283, 1989-   2. Patterson, L. H. Rationale for the use of aliphatic N-oxides of    cytotoxic anthraquinones as prodrug DNA binding agents: a new class    of bioreductive agent. Cancer and Metastasis Revs. 12, 119-134,    1993.-   3. Patterson, L H, Craven, M R, Fisher, G R and Teesdale-Spittle, P.    Aliphatic amine N-oxides of DNA binding agents as bioreductive    drugs. Oncology Research 6, 533-538, 1994.-   4. Mckeown, S R, Hejmadi, M V, McIntyre, I A, McAleer, J J A and    Patterson, L H. AQ4N: an alkylaminoanthraquinone N-oxide showing    bioreductive potential and positive interaction with radiation in    vivo. Brit J Cancer, 72,76-81.-   5. Mckeown, S R, Hejmadi, M V, McIntyre, I A, McAleer, J J A and    Patterson, L H. AQ4N: an alkylaminoanthraquinone N-oxide showing    bioreductive potential and positive interaction with radiation. Brit    J Cancer, 72, 76-81, 1995.-   6. Wilson, W R, Denny, W A, Pullen, S M, Thompson, K M, Li, A E,    Patterson, L H. Tertiary amine N-oxides as bioreductiver drugs: DACA    N-oxide, nitracrine N-oxide and AQ4N, Drit J Cancer, 74, 543-47,    1996.-   7. McKeown, S R, Friery, O P, McIntyre, I A, Hejmadi, M V, Patterson    L H. Evidence for a therapeutic gain when AQ4N or tirapazamine is    combined with radiation Brit J Cancer 74, S39-42, 1996-   8. Hejmadi, M V, McKeown, M V, Priery, O P, McIntyre, I A,    Patterson, L H and Hirst, D G. DNA damage following combination of    radiation with the bioreductive drug AQ4N: possible selective    toxicity to oxic and hypoxic cells. Brit J Cancer, 73, 499-505,    1996.-   9. Smith, P J, Blunt, N J, Desnoyers, R, Giles, Y and Patterson,    L H. DNA topoisomerase II dedendent cytotoxicity of    alkylaminoanthraquinones and their N-oxides. Cancer Chemotherap.    Pharmacol, 39, 455-461 (1997)-   10. Smith, P J, Desnoyers, R, Blunt, N, Giles, Y and Patterson, L H.    Flow cytomeric analysis and confocal imaging of anticancer    alkylaminoanthracuinones and their N-oxides in intact human cells    using 647 nm Krypton laser excitation. Cytometry, 27, 1, 43-53,    1997.    CLSM Imaging of Dual Fluorochrome Vital Cell Staining

We have sought to demonstrate the spectral separation of the DRAQ5fluorescence signal from that of other commonly used fluorochromes byusing selective excitation. FIG. 3 b shows the significant variation inthe capacity of the A375 cells for intracellular conversion of FDA byesterase cleavage to the retained form of fluorescein. Imaging the samesample using selective excitation of DRAQ5 clearly demonstrates nuclearmorphology (FIG. 3 c), while transmission imaging (FIG. 3 a) revealsoverall cellular form. Triple imaging analysis identifies daughter cellpairs (marked x, y and z by arrows in FIG. 3 d). Dual imaging wasextended to a vital dye capable of defining cytomplasmic organelles.FIG. 4 a-c shows DRAQ5 (nuclei) and rhodamine 123 (mitochondria)co-labelled cells.

Thus, FIGS. 3 a-d show differential excitation of fluorescein and DRAQ5in viable A375 cells analysed by CLSM. Cells were treated with 10 μMDRAQ5×1 h and subsequently labelled with FDA at 1 μM for 15 min. Panelsshow the same view imaged as follows: a, transmission image; b 488 nmexcitation of fluorescein; c, 647 nm excitation of DRAQ5; d, mergedimages of a-c encoded blue, green and red respectively. Images are250×250 μm; pairs of daughter cells are indicated by arrows.

FIGS. 4 a-c show images a-c showing the differential excitation ofrhodamine 123 and DRAQS in viable A375 cells analysed by confocal laserscanning microscopy. Cells were treated with 10 μM DRAQ5×1 h andsubsequently labelled with rhodamine 123 at 2 Ag/ml for 5 min. Images aand b show the same view with either 488 nm or 647 nm excitationrespectively. Image c represents the merged images of a (encoded green)and b (encoded red). Images are 1.00×100 μm.

EXAMPLE 5 Flow Cvtometry Applications of DRA5S as a Novel Deep Red/InfraRed Fluorescent DNA-Binding Probe

Flow cytometry, as used here, is a process for the measurement of thelight scatter and fluorescence characteristics of cells or particlespassing through a measuring apparatus in a fluid stream in which singlecells traverse the focus position(s) of single or multiple laser beams.The time delay in passing through spatially separated focus positions ismonitored electronically allowing the cytometer to generate fullycorrelated multioarameter measurements for multibeam configurations,Here we demonstrate the use of DRAQ5 in single, dual and triple beamsystems in a set of applications using human cells.

Cell Culture

HL60 (human promyelocytic-leukaemia cell line) and SUD4 (human B celllymphoma cell line) were grown as suspension cultures-in-RMPI mediumwith 10% foetal calf serum, 1 mM glutamine and antibiotics and incubatedat 37° C. in an atmosphere of 5% CO₂ in air. For flow cytometryexperiments, asynchronously growing suspension cultures were diluted to2.5-4>10⁵ cells/ml at 2 h prior to drug treatment. Cell cycle-perturbedpopulations were obtained by treating SUD4 cells with the drug etoposide(VP-16-213) at 0.25 uM for 18h. Cells were treated with DRAQ5 and FDA asdescribed above. Cell concentrations were determined using a Coultercounter and cell cycle distribution determined using an algorithm forthe normal distibution of fluorescence intensity profiles forfluorochrome stained G1 and G2 cells.

Suspension cultures were analysed by flow cytometry without washing.Human blood was obtained using routine venepuncture of a healthy donorand samples manipulated using standard haematological procedures for theisolation of mononuclear blood cells and surface antigen recognitionusing antibody panels (see Table 1). TABLE 1 FACScan ™ (Cytometer C)flow cytometric analysis of DRAQ5-labelled human cells Cell prepara-tion & Mean fluorescence intensity (±sd) DRAQ5 of gated population ^(a):exposure Lympho- Granulo- (min) SUD4 cells cytes Monocytes cytesPreparation 1: viable cultured cells ^(b) 0 1.3 ± 6.8 5 499.4 ± 143.3 5532.4 ± 148.3 120  645.1 ± 177.7 0  6.0 ± 16.6 (0.25 μM VP-16) 5 891.8 ±126.4 (0.25 μM VP-16) Preparation 2: viable cultured cells, surfaceantigen analysis ^(c) 0  5.2 ± 13.8 5 564.3 ± 147.2 0 13.4 ± 21.6 (0.25μM VP-16) 5 902.7 ± 117.1 (0.25 μM VP-16) Preparation 3: Ficoll gradient-isolated viable mononuclear blood cells ^(d) 0  0.8 ± 4.6  1.7 ± 5.7 5227.4 ± 24.5 302.9 ± 25.4 5 215.9 ± 26.3 301.8 ± 24.3 (4.8 × 10⁵/ml) 5234.1 ± 27.0 311.0 ± 30.9 (7.5 × 10⁵/ml) 120  299.1 ± 23.9 342.2 ± 22.4Preparation 4: Preparation 3 plus surface antigen analysis ^(e) 0  0.0 ±0.1  0.0 ± 0.1 5 261.6 ± 26.3 317.9 ± 27.3 Preparation 5: Whole blood,viable cells surface antigen analysis ^(f) 0  0.0 ± 0.2  0.1 ± 1.1  0.1± 2.1 5 257.3 ± 35.0 274.6 ± 41.0 248.4 ± 35.9 Preparation 6:Preparation 5 but cells lysed and fixed ^(g) 0  0.0 ± 0.3  0.0 ± 0.9 0.1 ± 3.0 5 228.5 ± 28.5 240.2 ± 28.1 254.3 ± 31.1Footnotes to Table^(a) Fluorescence detected on FL3 and analysed as pulse area parameterfor cell populations gated on the relevant forward-scatter (FSC) andside-scatter (SSC) characteristics.# All cell preparations analysed at 2.5 × 10⁵/ml, unless otherwiseindicated, in phosphate buffered saline plus 1% BSA # (ie analysisbuffer) without (0 min) or with (5 or 120 min exposure) 20 μM DRAQ5.^(b) Preparation 1: Cells derived from cell culture of the SUD4 humanfollicular B cell lymphoma line and resuspended in analysis buffer.Parallel analysis of samples using# conventional ethidium bromide staining, of RNase A-digestedpermeabilised cells yielded G1 = 34.9%, S phase = 48.0%, G2/M = 17.1%for # asynchronous cultures, and G1 = 0.6%, S phase = 55.3%, G2/M =44.0% for late cell cycle-arrested cells obtained by treating cells withthe cytotoxic drug VP-16 # (0.25 μM × 18 h). Quantitative analysis ofDNA content of G1 (SUD4): G1 (normal diploid lymphocytes) gave a ratioof 1.083.^(c) Preparation 2: As for Preparation 1 but processed for surfaceantigen analysis using directly labelled antibodies: anti-CD54-FITC(detected on FL1 parameter), anti-CD19-PE# (detected on FL2 parameter).^(d) Preparation 3: Ficoll gradient-separated viable mononuclear bloodcells from normal donor. Sample obtained by routine venepuncture (ratiolymphocytes:monocytes = 11.5:1)# and resuspended in analysis buffer.^(e) Preparation 4: As for preparation 3 but processed for surfaceantigen analysis using anti-CD45-FITC.^(f) Preparation 5: Whole blood from normal donor processed for surfaceantigen analysis, analysed as viable cells using anti-CD45-FITCpositivity as the FL1 parameter master trigger to exclude RBCs. Gatedpopulations of 25.5% lymphocytes, 13.3% monocytes, 61.2% granulocytes^(g) Preparation 6: As for preparation 5 but after processing forsurface antigen analysis, cells fixed and RBCs lysed in FACSLyse ™ andre-suspended in analysis buffer. FSC parameter as master trigger. Gatedpopulations of 43.7% lymphocytes, 9.7% monocytes, 46.6% granulocytes.Flow Cytometry

Cells were analysed using one of four flow cytometers according to theexcitation requirements.

Cytometer A: Single beam high power 647 nm krypton laser excitation: Thesystem was a custom-built cytometer and incorporated an Innova 3000Kkrypton laser (Coherent Corp., Palo Alto, Calif., USA) tuned to the 647nm line. Forward light scatter, 900 light scatter and fluorescenceemissions were collected for 1×10⁴ cells using the 90° light scatterparameter as the master signal. The optical system permitted theanalysis of various fluorescence emission wavelengths including: >715 nm(termed low infra-red) and, as reported here >780 nm fluorescence(infra-red ). Forward and 90° light scatter were analysed for theidentification of cell debris. Laser power was set at 200 mW and linearamplifiers were used for the fluorescence signals. The analysis opticsincluded a 675 nm cold dichroic mirror, ambient laboratory temperaturewas approximately 12° C. and the sheath reservoir was maintained at 10°C. Filters were supplied by Melles Griot. Median, mean and modeparameters were calculated for the distribution of fluorescenceintensity-values throughout a given cell population. In all experiments,median and mean values produced very similar results. Median valuesalone are reported since this parameter is less affected by the presenceof highly fluorescent cells beyond the upper limit for quantification.

Cytometer B: Dual beam low power 633 nm/high power 488 nm laserexcitation: The system was a FACS 440 cell sorter (Becton DickinsonInc., Cowley, UK) incorporating a Spectra Physics argon ion laser (max500 mW output), tuned to the 488 nm line (100 mW output), and asecondary Spectra Physics 156 helium-neon laser emitting at 633 nm(emitting <5 mW), with a temporal beam separation of about 30 μsec.Forward light scatter, 90° light scatter and fluorescence emissions werecollected for 1×10⁴ cells using the forward light scatter parameter asthe master signal from the primary 488 nm beam, while side scatter wascollected through a 488/10 nm band-pass filter. The analysis opticsincluded: i) a cold dichroic mirror (transmitting >675 nm), ii)fluorescence from fluorescein excited by the 488 nm beam detected at aPMT guarded by a 535/15 nm band-pass filter with no signal delay, andiii) a red-sensitive PMT with an appropriate delay, additionally guardedby a 620 nm long-pass filter, to detect the transmitted beam ofDRAQ5-associated fluorescence at wavelengths beyond 675 nm (high-red andextending into the infra red region of the spectrum) Forward and 90°light scatter were analysed to exclude any cell debris. All parameterswere acquired at 256 channel resolution with Consort 30 software (BectonDickinson) and subsequently analysed with WinDI software (J. Trotter, LaJolla, Calif.). The system employed the same analysis optics when usedin the single 488 nm beam mode but with no signal delay for thered-sensitive PMT.

Cytometer C: Single beam, low power 488 nm laser excitation: The systemwas a FACScan (Becton Dickinson; Inc., Cowley, UK) incorporating anargon ion laser (max 15 mW output), tuned to the 488 nm line. Forwardlight scatter, 90° light scatter and fluorescence emissions werecollected for 1×10⁴ cells using the forward light scatter parameter asthe master signal. The standard analysis optics provided the FL1(blue)/FL2 (green)/FL3 (red) PMT parameters with pulse analysisperformed on the FL3 originating signals.

Cytometer D: Triple beam medium power 633 nm/medium power 488 nm/mediumpower multiline-UV laser excitation:

The system was a FACS Vantage cell sorter (Becton Dickinson Inc.,Cowley, UK) incorporating a Coherent Enterprise II laser simultaneouslyemitting at multiline UV (350-360 nm range) and 488 nm wavelengths withthe beams made non-colinear using dichroic separators. Beam-combiningoptics were used to align the UV beam with that emitted by a SpectraPhysics 127-35 helium-neon laser (max 35 mW output) emitting at 633 nmwith a temporal separation of about 25 μsec from that of the primary 488nm beam. Forward light scatter, 90° light scatter and fluorescenceemissions were collected for 1×10⁴ cells using the forward light scatterparameter as the master signal from the primary 488 nm beam, while sidescatter was collected through a 488/10 nm bandpass filter. The analysisoptics were: i) primary beam-originating signals analysed at FL1 (FITCfilter; barrier filter of 530/30 nm) after transmission at SP610 andSP560 dichroics, or at FL2 (barrier filters of 585/42 nm or 575/26 nm)after transmission at SP610 and reflection at SP560 dichroics, or at FL3(barrier filter of LP715 nm) after reflection at a SP610 dichroic; ii)delayed beam-originating signals analysed at FL4 (barrier filter ofLP695 nm) or at FL5 (barrier filter of DF424/44 nm) after transmissionor reflection at a LP640 dichroic respectively. Forward and 90° lightscatter, were analysed to exclude any cell debris. All parameters wereanalysed using CellQuest software (Becton Dickinson).

Whole Cell Fluorescence Detected by Flow Cytometry

Despite DRAQ5 excitation being optimal at the 647 nm laser wavelength,preliminary studies indicated that the probe could be sub-optimallyexcited at lower wavelengths, including multi-line UV 488 nm, 514 nm and633 nm. Here we have sought to assess DRAQ5 as a DNA probe for use inflow cytometry by comparing the four different cytometer configurations:

Cytometer A: Single-beam high power 647 nm krypton laser excitation.

Cytometer B: Dual-beam low power 633 nm/high power 488 nm laserexcitation.

Cytometer C: Single-beam low power 488 nm laser excitation.

Cytometer D Triple-beam medium power 633 nm/medium power 488 nm/mediumpower multiline-UV laser excitation.

FIG. 5 shows that using a low power HeNe laser (Cytometer B), completeseparation does not occur for autofluorescence and DRAQ5 signals forviable HL-60 cells treated with a low, non-saturating DRAQ5concentrations. Further studies (data not shown) indicate that completeseparation could be achieved after a two hour incubation with 20 μMDRAQ5. However even under these limiting excitation conditions the 633nm derived DRAQ5 signal shows a clear linear dose-response (see inset toFIG. 5) down to approximately 2.5 μM, comparable with the linearityobtained for optimal 647 nm excitation (using Cytometer A) and detectionat wavelengths >780 nm.

FIG. 6 a and b show that a low power HeNe laser (Cytometer B) can beused to identify DRAQ5-associated fluorescence in fluorescein-loadedcells analysed in a dual beam configuration. FIG. 6 c and d showsco-excitation of DRAQ5 and fluorescein is possible using a single beamof 488 nm wavelength (Cytometer B). There is clear separation ofsignals, due to the distinct, non-overlapping spectra, despite the lowintensity signal derived from sub-optimal excitation of DRAQ5.

We have sought to demonstrate the utility of DRAQ5 in a single beamcytometer (ie FACScan™; Cytometer C). FIG. 7 a-d shows typical resultsdemonstrating the ability of DRAQ5 to identify nucleated cells incomplex populations. FIG. 7 a shows the detection of cell cycledistribution-versus cell surface antigen expression for intact cells.FIG. 7 b shows the discrimination of subsets according to stainingpotential while FIGS. 7 c and 7 d demonstrate the application of DRAQ5in detecting nucleated cells in whole blood and lysed blood. Factorsrelating to the ability of DRAQ5 to stain nuclei are analysed in theTable. Using viable cultured. asynchronous cells (Preparation 1) DRAQ5rapidly stained cells in a reproducible manner and generatedfluorescence distinct from the autofluorescence background. The large sdvalues derive from the spread of cells throughout the cell cycle. Themean value reflects mean cellular DNA content as evidenced by the1.7-fold increase for G2 arrested populations. The processing of cellsfor surface antigen analysis (Preparation 2) does not affect the abovecharacteristics. The isolation of intact mononuclear blood cells(Preparations 3 and 4) yields samples which can be stained within aconvenient cell density range and be processed for surface antigenanalysis. In Preparations 3 and 4 we have consistently observed anenhanced staining potential of monocytes versus lymphocytes (1.14-1.4fold) indicating that viable cell staining potential may be used as afactor for subpopulation discrimination. The results for whole bloodshow that nucleated cells (including granulocytes) can be stained to asimilar degree in the presence (Preparation 5) of red blood cells (RBCs)or following RBC lysis and mild fixation (Preparation 6).

FIG. 8 summarises the DRAQ5 concentration-dependent differences in DRAQ5staining for viable cell populations obtained using Preparation methods1 and 2 (see footnote to Table). The populations show similar titrationcurves with saturation occurring in a manner which reflects relative DNAcontent (for a given cell type, eg SUD4) or nuclear staining potential(eg lymphocytes versus monocytes) at concentrations of ≧10 μM.

FIG. 9 demonstrates the utility of DRAQ5 for the detection of the cellcycle specific expression of an intracellular protein, in fixed cells,detected using fluorochrome-tagged antibodies activated by 488 nm (FITC)and multiline-UV wavelengths (Cytometer D). FIGS. 10 a-f show that in atriple beam configuration (Cytometer D) it is possible to demonstrateDRAQ5 fluorescence activated by two separate beams with discriminationon a third for the monitoring of relatively rare cell cycle events suchas high cyclin B1 expression in G2/M of asynchronous cultures.

Thus, FIG. 5 shows flow cytometric analyses of DRAQ5 accumulation, for aone hour exposure period, in viable HL60 cells. Frequency distributionhistograms are for 1 cw power 633 nm wavelength excitation usingCytometer B. Symbols: ∘, ▴ and ▪ represent 0, 5 and 10 μM DRAQ5respectively. Inset: Linearity of DRAQ5 dose-response, using twodifferent Cytometers (namely B and A with correlation coefficients of0.96 and 0.97 for 633 nm and 647 nm excitations respectively).

FIGS. 6 a to d show dual beam flow cytometric analysis (Cytometer B) forthe detection of DRAQ5-associated fluorescence in fluorescein-labelledviable HL60 cells. Representative flow cytometric bivariate plots ofgreen (FL2-height; fluorescein) versus deep red/low infra red(FL1-height; DRAQ5) whole cell fluorescence signals. Panels a and b showdual beam excitation of fluorescein (488 nm) and DRAQ5 (low power 633nm). Panel: a, FDA (0.2 μM for 10 min alone); b, cell pretreated with 5μM DRAQ5 for 1 h prior to FDA treatment. Panels c and d repeat the samecell treatment conditions except for the use of single beam excitationat 488 nm for fluorescein and DRAQ5. Numbers indicate the percentage ofgated events within the quadrant regions.

FIGS. 7 a to d show single beam flow cytometric analysis of (CytometerC) of DRAQ5 fluorescence (FL3-area) versus antibody fluorescence(FL2-height monitoring phycoerythrin-labelled anti-CD19 or FL1-heightmonitoring FITC-labelled anti-CD45) for cultured and blood-derived humancells. Cell suspensions (2.5×10⁵/ml) were maintained in phosphatebuffered saline containing 1% bovine serum albumin. Human bloodmononuclear cell subpopulations, obtained using standard Ficoll gradientseparation, were identified and gated according to their forward- andside-light scatter characteristics. Doublets were excluded by pulseanalysis gating on normal FL3-area versus FL3-width parameter values.Panel a: cultured asynchronous SUD4 lymphoma cells. Panel b: bloodmononuclear cell subpopulations, obtained using standard Ficoll gradientseparation. Panel c: whole blood (triggered on CD45+ events). Panel d:lysed whole blood. Arrowed subpoulations: G1, S and G2/M represent cellcycle phases; L, lymphocytes; M, monocytes; G, granulocytes; N, nucleilacking plasma membranes.

FIG. 8 shows single beam low cytometric quantification (Cytometer C) offluorescence intensity of cultured and blood-derived human cells exposedto DRAQ5 at room temperature for 5 min. Data are mean values (±SD) andrepresent results from a typical experiment. Symbols: ∘, culturedasynchronous SUD4 lymphoma cells; □, SUD4 cells exposed to 0.25 μM VP-16for 18 h to arrest cells in S phase and G2 of the cell cycle; ●lymphocytes; ▪, monocytes.

FIG. 9 shows dual beam flow cytometric analysis (Cytometer D) of thecell cycle specific expression of cyclin B1. Fixed, RNaseA-digested andDRAQ5-stained (FL3; 488 nm excitation) SUD4 cells were obtained from anasynchronous culture exposed to 0.25 μM VP-16 for 18 h to accumulatecells in G2/M. G2/M phase-expressed cyclin B1 protein was monitored byindirect immunofluorescence using AMCA-labelled second antibody(FL5-height; multiline UV excitation) to detect the binding ofanti-cyclin B1 (GNS1) mouse monoclonal IgG. Panels a and c show antibodycontrols (non-specific IgG plus second antibody). Panels b and d showresults for specific antibody plus second antibody. Antibodies wereobtained from Santa Cruz Biotechnology Inc. Arrowed subpoulations: G1, Sand G2/M represent cell cycle phases; unlettered arrow shows expectedposition of cells expressing high levels of cyclin B1 and located inG2/M of the cell cycle.

FIGS. 10 a to f show triple beam flow cytometric (cytometer D) analysisof DRAQ5-stained fixed and RNase A digested asynchronous SUD4 lymphomacells. DRAQ5 fluorescence (pulse height) monitored by FL3 (488 nmexcitation) and FL4 (633 nm excitation). Cell cycle-independent Cdc2protein and the G2/M phase-expressed cyclin B1 protein were monitored byindirect immunofluorescence using FITC-labelled second antibody(FL1-height; 488 nm excitation) to detect the binding of anti-Cdc2 p34(H-297) rabbit polyclonal IgG, and AMCA-labelled second antibody(FL5-height; multiline UV excitation) to detect the binding ofanti-cyclin B1 (GNS1) mouse monoclonal IgG. Antibodies were obtainedfrom Santa Cruz Biotechnology. Panels: a and b, DNA versus Cdc-2 p34; cand d, DNA versus cyclin B1; e and f, DNA histograms for blue and redexcitation wavelengths respectively. Arrowed subpopulations: G1, S andG2/M represent cell cycle phases; HCyB, high cyclin B1 expressing cellslocated in G2/M of the cell cycle.

Flow Cytometric Analysis of Multi-Line UV Excitation of DRAQ5 StainedCells

The absorbance peaks noted for wavelengths <400 nm suggest thatchromophore excitation at near UV wavelengths should be possible (datanot shown). It has been demonstrated that DRAQ5-stained nuclei of livingcells can be excited in the near-UV region of the spectrum as shown bythe use of multi-line UV flow cytometry (Cytometer D; FIG. 11 a-c).Although UV-excitation is less efficient than at 647 nm wavelength (FIG.11 b) and detection require increased photomultiplier signalamplification, fluorescence intensities clearly reflect cellular DNAcontent distribution (FIG. 11 c). This demonstrates that in triple beamcombinations, DRAQ5 can provide a DNA discriminating signal derived fromUV, and visible range excitation wavelengths.

FIGS. 11 a-c show the 488 nm (panel a), 647 nm (panel b), or multi-lineUV (350-360 nm range; panel c) excitation of DRAQ5 in intact SUD4lymphoma cells for emission at >695 nm wavelengths and analysed bymulti-beam flow cytometry (Cytometer D). Bold lines reflect the cellularDNA content of DRAQ5-stained cells; feint lines represent non-stainedcontrol cells; dotted lines represent reference allophycocyanine-(APC)stained reference micro-beads used as 647 nm excitable standards.

EXAMPLE 6 Differential Cellular Accumulation of an N-Oxide Derivative ofDRAQ5 (DRAQ5NO) in the Discrimination of Intact and Dead Cells

The ability to discriminate intact viable cells from those undergoingthe various stages of cell death can be achieved through thedifferential cellular accumulation of chemical probes including certainfluorochromes. A particular type of cell death, termed apoptosis, hasdiscernible early stages which can occur in intact cells. Discriminationis used extensively in both biological and clinical assays. For exampleflow cytometric assays may allow for the identification, quantification,analysis, preparation or exclusion of cell subsets. Probe uptake andretention is dependent upon multiple factors, including the integrity ofthe plasma membrane (eg affecting probe entry) arid the intracellularbehaviour of the probe (eg probe binding to nuclear DNA). Currentfluorometric assays for cell death can use the ability of intact viablecells to remain unstained by excluding the probe (eg the fluorescent DNAstain prodidium iodide), while cells with compromised membranes allowaccess of the probe to nuclear DNA. Cells undergoing the early stages ofapoptotic cell death can be identified by the cell surface binding ofthe fluorochrome-tagged chemical, Annexin V, but show no loss ofmembrane integrity. The later stages of cell death and apoptosis, whenthe plasma membranes become disrupted, are associated with high AnnexinV-binding and high propidium iodide DNA-staining.

Here we exemplify the use of an N-oxide derivative of DRAQ5 (DRAQ5NO)providing an enhancement to live-dead cell discrimination. DRAQ5NO iscapable of entering into and being retained by intact viable cells at alow level, providing a positive discrimination for intact cells. Incombination with a secondary probe (eg Annexin V) there is enhanceddiscrimination of the stages in the progression of cells through theprocess of cell death or apoptosis. The four stages, according to thestaining patterns are:

-   stage 1: DRAQ5NO positive/Annexin V negative (intact viable cells)-   stage 2: DRAQ5NO positive/Annexin V positive (early stage apoptotic    cells)-   stage 3: DRAQ5NO high positive/Annexin V positive (late stage    apoptotic/dead cells)-   stage 4: DRAQ5NO negative/Annexin V positive (non-nucleated cellular    debris)

FIG. 12 a-d and FIG. 13 a-d illustrate examples, using a human B celllymphoma cell line capable of progression through apoptosis in responseto the anticancer drug VP-16 (etoposide) for an 18 h exposure to 0.25μM. Cells were prepared by standard methods for Annexin V-FITC binding,simultaneously exposed to 50 μM DRAQ5NO and then diluted 1:5 inphosphate buffered saline prior to flow cytometric analysis usingCytometer D. The instrument was used in a dual laser mode with 488 nmwavelength excitation of FITC (monitored by parameter FL1-H) and 633 nmwavelength excitation of DRAQ5NO (monitored by parameter FL4-H) FIG. 12a-d show the combinations of reagent treatments (Anx=Annexin V-FITC;AQ5N=the N-oxide derivative of DRAQ5) for control cells and FIG. 13 a-dshow the same combination of reagents for VP-16 (i.e. VP) treatedcultures. The results show the low level of DRAQ5NO staining achieved instage 1 populations and the increased level in stage 3 cells. Thefrequency of cells which are Annexin V positive is increased by VP-16treatment but comprise three populations (stages 2-4) discernible usingthe quadrant analysis shown in the plots. The enhancement provided bythe use of DRAQ5NO is with respect to two features. First, theadvantageous spectral properties of the DRAQ5 derivative allowing forthe separation of the probe excitation events by the use of two lasersand/or the greatly reduced spectral overlap of the probe emissionsignals. Second, the positive discrimination of intact cells fromnon-nucleated cellular debris.

1. An isolated compound of the following formula:

wherein each of X₁ and X₂ are independently NH-A-NR¹R², and wherein A isa C₂₈ alkylene group and R¹ and R²are independently selected fromhydrogen, C₁₋₄ alkyl, C₂₋₄ hydroxyalkyl and C₂₋₄ aminoalkyl, or R¹ andR² together form a C₂₋₆ alkylene group which with the nitrogen atom towhich R¹ and R² are attached forms a heterocyclic ring.
 2. A compoundaccording to claim 1, wherein, when R¹ and R² form a heterocyclic ring,the ring has 3 to 7 atoms therein.
 3. A compound according to claim 1,wherein X₁ and X₂ Are NH(CH₂)₂NR¹R².
 4. A compound according to claim 1,wherein R¹ and R² are a C₁₋₄ alkyl.
 5. A compound according to claim 1,wherein R¹ and R² are methyl.
 6. An aqueous composition comprising acompound according to claim
 1. 7. An isolated compound of the followingformula:

wherein each of X₁ and X₂ are independently NH-A-NR¹R², and R¹ and R²arehydrogen.
 8. A compound according to claim 7, wherein, when R¹ and R²form a heterocyclic ring, the ring has 3 to 7 atoms therein.
 9. Acompound according to claim 7, wherein X₁ and X₂ are NH(CH₂)₂NR¹R². 10.A compound according to claim 7, wherein R¹ and R² are a C₁₋₄ alkyl. 11.A compound according to claim 7, wherein R¹ and R² are methyl.
 12. Anaqueous composition comprising a compound according to claim
 2. 13. Anaqueous composition comprising a compound according to claim
 2. 14. Anaqueous composition comprising a compound according to claim
 3. 15. Anaqueous composition comprising a compound according to claim
 4. 16. Anaqueous composition comprising a compound according to claim 5.